The following definitions are provided to facilitate an understanding of the present invention. The term "biological binding pair" as used in the present application refers to any pair of molecules which exhibit natural affinity or binding capacity. For the purposes of the present application, the term "ligand" will refer to one molecule of the biological binding pair and the term "antiligand" or "receptor" will refer to the opposite molecule of the biological binding pair. Two complementary strands of nucleic acid are biological binding pairs. One of the strands is designated the ligand and the other strand is designated the antiligand. However, biological binding pairs may also comprise antigens and antibodies, drugs and drug receptor sites and enzymes and enzyme substrates.
The term "probe" refers to a ligand of known qualities capable of selectively binding to a target antiligand. As applied to nucleic acids, the term "probe" refers to a strand of nucleic acid having a base sequence complementary to a target base sequence. Typically, the probe is associated with a label to identify a target base sequence to which the probe binds, or the probe is associated with a support to bind to and capture a target base sequence. The term "primer" is used to refer to nucleic acid having a base sequence complementary to a target base sequence, which upon nucleic acid hybridization is used to promote a reaction. These reactions usually involve enzymes called polymerases and transcriptases.
The term "label" refers to a molecular moiety capable of detection including, by way of example, without limitation, radioactive isotopes, enzymes, luminescent agents, dyes and detectable intercalating agents. The term "agent" is used in a broad sense, in reference to labels, and includes any molecular moiety which participates in reactions which lead to a detectable response. The term "cofactor" is used broadly to include any composition which participates in reactions with a label agent.
The term "support" refers to conventional supports such as beads, particles, dipsticks, fibers, filters, membranes and silane or silicate supports such as glass.
The term "amplify" is used in the broad sense to mean creating an amplification product which may include, by way of example, additional target molecules, or target-like molecules or molecules complementary to the target molecule, which molecules are created by virtue of the presence of the target molecule in the sample. In the situation where the target is a nucleic acid, an amplification product can be made enzymatically with DNA or RNA polymerases or transcriptases.
Genetic information is stored in living cells in threadlike molecules of deoxribonucleic acid (LNA). In vivo, the DNA molecule is a double helix, each strand of which is a chain of nucleotides. Each nucleotide is characterized by one of four bases: adenine (A), guanine (G), thymine (T), and cytosine (C). The bases are complementary in the sense that due to the orientation of functional groups certain base pairs attract and bond to each other through hydrogen bonding. Adenine in one stand of DNA pairs with thymine in an opposing complementary stand. Guanine in one strand of DNA pairs with cytosine in an opposing complementary strand. In ribonucleic acid (RNA), the thymine base is replaced by uracil (U) which pairs with adenine in an opposing complementary strand.
DNA consists of covalently linked chains of deoxribonucleotides and RNA consists of covalently linked chains of ribonucleotides. The genetic code of a living organism is carried upon DNA in the sequence of the base pairs. Proteins are made or expressed by living organisms in a process in which a DNA sequence is transcribed to a RNA sequence and the RNA sequence translated into proteins.
Each nucleic acid is linked by a phosphodiester bridge between the five prime hydroxyl group of the sugar of one nucleotide and the three prime hydroxyl group of the sugar of an adjacent nucleotide. Each linear strand of naturally occurring DNA or RNA has one terminal end having a free five prime hydroxyl group and another terminal end having a three prime hydroxyl group. The terminal ends of polynucleotides are often referred to as being five prime (5') termini or three prime (3') termini in reference to the respective free hydroxyl group. Complementary strands of DNA and RNA form antiparallel complexes in which the 3' terminal end of one strand is oriented to the 5' terminal end of the opposing strand.
Nucleic acid hybridization assays detect the tendency of pairs of nucleic acid strands to pair with greatest stability if they contain regions of complementary sequence. Each pair of complementary nucleotides, between two strands, increases the stability of pairing between a biological binding pair formed between the two nucleic acids. DNA segments isolated from a growing organism are generally duplex DNA, a pair of perfectly complementary strands whose pairing is very stable. The term "hybridize" refers to imposing conditions which promote such pairing. The term "denature" refers to imposing conditions which discourage such pairing. These conditions are imposed by adjusting ionic strength, pH or temperature.
Polymerases and transcriptases are enzymes which, in the presence of appropriate reaction conditions, produce complementary copy of a strand of DNA or RNA. The strand that is copied is called the template DNA or RNA.
A polymerase chain reaction, PCR, uses a pair of nucleic acid primers to synthesize copies of target nucleic acid. One primer hybridizes to a target sequence on a first strand, and a second primer hybridizes to a second target sequence on the second strand. This permits the amplification product directed by one of the pair of primers to serve as a template for synthesis directed by the second member of the pair of primers. PCR is carried out using a solution containing both members of the pair of primers and a polymerase capable of withstanding conditions required to denature paired strands of DNA.
The identification of unique DNA or RNA sequence or specific genes within the total DNA or RNA extracted from tissue or culture samples may indicate the presence of physiological or pathological conditions. In particular, the identification of unique DNA or RNA sequences or specific genes, within the total DNA or RNA extracted from human or animal tissue, may indicate the presence of genetic diseases or conditions such as sickle cell anemia, tissue compatibility, cancer and precancerous states, or bacterial or vital infections. The identification of unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from bacterial cultures or tissue containing bacteria may indicate the presence of antibiotic resistance, toxins, viruses, or plasmids, or provide identification between types of bacteria.
Thus, nucleic acid hybridization assays have great potential in the diagnosis and detection of disease. Further potential exists in agricultural and food processing where nucleic acid hybridization assays may be used to detect plant pathogenesis or toxin-producing bacteria.
Much research is presently directed to identifying the nucleic acid sequences which define organisms. An initial step in the process is the identification of regions within the nucleic acid, a process known mapping. These regions may be subjected to further sequencing. Both the mapping process and the sequencing process are slow and tedious.